Adipose Differentiation Related Protein (ADFP), VLDL Biogenesis and Triacylglycerol Homeostasis Nonalcoholic fatty liver disease (NAFLD) is often a precursor to chronic liver diseases, and a predictor of cardiovascular disease. The hallmark of NAFLD is the presence of numerous lipid droplets (LDs) in the hepatocytes. Adipose differentiation related protein (ADFP) is the most abundant cytosolic protein that binds to the surface of these LDs. Its expression is upregulated in lipid infiltrated hepatocytes (in NAFLD). I generated ADFP deficient mice and found a 60% reduction in hepatic triacylglycerol (TG) in these mice compared to WT mice. The TG reduction is pronounced in the cytosol, but, paradoxically, TG is increased in the microsome fraction in ADFP deficient as compared with wildtype mice. Concomitantly, there is an increase in the amount of microsomal triglyceride transfer protein (MTP) at the protein level, but not mRNA level, and a increase in VLDL secretion in the ADFP deficient mice. The hepatic lipid uptake, production and utilization are not altered. In this application, I will pursue three specific aims: (1) to identify the molecular mechanism of ADFP deficiency-associated hepatic MTP protein upregulation, (2) to determine the interplay between MTP and ADFP in modulating hepatic TG homeostasis., and (3) to study Adfp deficiency in the heart. I will use hepatic cell lines, cardiomyocytes, primary hepatocytes, transformed Adfp deficient hepatic cells, ADFP deficient and MTP conditional knockout mice as models. I will also generate conditional Adfp targeting mice. I will apply a combination of pharmacological and molecular tools such as MTP inhibitor, siRNA and recombinant helper dependent adenovirus to suppress or augment gene expression in these model cells and animals. The ultimate objectives I want to achieve are (i) to determine the mechanism of MTP protein upregulation in ADFP deficiency, (ii) to find the mechanism of ADFP deficiency-associated hepatic TG reduction and cardiac TG elevation, (iii) to test the hypothesis that ADFP, with its involvement in LD formation and regulation may represent a cytosolic protein which compete with MTP for TG and negatively regulate VLDL secretion while causing hepatic steatosis when it is overexpressed, and (iv) to identify factors that affect liver and heart TG storage in Adfp deficiency. My long term goal is to understand the function of ADFP and other LD- associated proteins in the formation and maturation of LD and how this affects the regulation of hepatic and cardiac TG dynamics such as lipoprotein assembly and secretion, and lipid homeostasis in liver heart and other organs.